IDENTIFICATION & CHARACTERIZATION OF TRICHOMONAD CYSTEINE PROTEASES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。滴虫寄生虫阴道毛滴虫导致人类最常见的非病毒性性传播感染之一，滴虫病。经亲和层析和Bio-Gel P-60柱层析分离得到的寄生虫和分泌型半胱氨酸蛋白酶(CP)组分，经细胞死亡检测ELISA(PLUS)试验和Annexin V-异硫氰酸荧光素流式细胞术分析表明，可诱导人阴道上皮细胞(HVEC)凋亡。细胞凋亡的启动与蛋白水解酶活性有关，因为特异性CP抑制剂E-抑制了两者的活性。对阴道毛滴虫分泌的半胱氨酸蛋白酶(CP)组分进行了SDS-PAGE和MS分析。CP组分的SDS-PAGE分析显示约30 kDa的三重峰，基质辅助激光解吸/电离飞行时间质谱仪显示两个密切相关的峰，分子量分别为23.6和23.8 kDa。对蛋白质消化的CP进行的质谱肽测序得到了与先前报道的克隆CP2、CP3和CP4(Mallinson，D.J.，Lockwood，B.C.，Coombs，G.H.和North，M.J.(1994)Microbitic 140,2725-2735)的匹配，以及另一个与CP4高度同源性的序列(www.tigr.org)。这两个物种是CP组分中含量最丰富的成分。这些结果提示，CP诱导的程序性细胞死亡可能参与了阴道毛滴虫体内感染的发病机制，这可能对治疗干预具有重要意义。这项研究结果最近发表在《传染病》杂志上。伊蒙。(72,4151-8,2004)和J.Biol.化学。(23853-60,2005年)

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The trichomonad parasite Trichomonas vaginalis causes one of the most common non-viral sexually transmitted infection in humans, trichomoniasis. The parasite as well as a secreted cysteine protease (CP) fraction, isolated by affinity chromatography followed by Bio-Gel P-60 column chromatography, were shown to induce apoptosis in human vaginal epithelial cells (HVEC) , as demonstrated by the Cell Death Detection ELISA(PLUS) assay and annexin V-fluorescein isothiocyanate flow cytometry analyses. Initiation of apoptosis was correlated with protease activity because the specific CP inhibitor E-64 inhibits both activities. The secreted cysteine protease (CP) fraction from T. vaginalis that was shown to induce apoptosis in HVEC was analyzed by SDS-PAGE and mass spectrometry. SDS-PAGE analysis of the CP fraction revealed triplet bands around 30 kDa, and matrix-assisted laser desorption/ionization time-of-flight MS indicated two closely associated peaks of molecular mass 23.6 and 23.8 kDa. Mass spectral peptide sequencing of the proteolytically digested CPs resulted in matches to previously reported cDNA clones, CP2, CP3, and CP4 (Mallinson, D. J., Lockwood, B. C., Coombs, G. H., and North, M. J. (1994) Microbiology 140, 2725-2735), as well as another sequence with high homology to CP4 (www.tigr.org). These last two species are the most abundant components of the CP fraction. These results, suggesting that CP-induced programmed cell death may be involved in the pathogenesis of T. vaginalis infection in vivo, may have important implications for therapeutic intervention. The results were recently published in Infect. Immun. (72, 4151-8, 2004 ) and J. Biol. Chem. (280, 23853-60, 2005)